Compositions of abscisic acid for animal health

ABSTRACT

The invention relates to compositions comprising abscisic acid, and/or salts, derivatives and analogs thereof, and methods of using the same for improving the health and survival of animal offspring.

FIELD OF THE INVENTION

The present invention generally relates to compositions comprisingabscisic acid, and/or salts, derivatives and analogs thereof, andmethods of their preparation and administration for and/or nutraceuticaluse in animals.

BACKGROUND OF THE INVENTION

Abscisic acid is a naturally occurring plant hormone and a safe,nontoxic substance. The chemistry and physiology of abscisic acid andits analogs is described by Milborrow, Ann. Rev. Plant Physiol. 1974,25, 259-307. The naturally occurring enantiomeric form of abscisic acidis (S)-(+)-abscisic acid. The stereochemistry of the side chain of themajor part of naturally occurring abscisic acid is 2-cis-,4-trans-,since that is the isomer that is produced biosynthetically by all greenplants and some microorganisms.

Certain salts of abscisic acid, and/or derivatives and analogs thereof,as described in U.S. application Ser. Nos. 12/011,846, 12/011,825,61/083,202, 61/083,203 and PCT/US08/01203, however, have demonstratedhigh concentrations of abscisic acid in their compositions, and areincorporated herein.

Commercial formulations comprising abscisic acid are used in agricultureand horticulture on or around crops and plants for improving stresstolerance, slowing the growth rate, and adjusting flowering phase.Abscisic acid has also been reported to possess insect inhibitionqualities. See U.S. Pat. Nos. 4,434,180 and 4,209,530. Others havereported potential medicinal properties of abscisic acid, for example USpatent application No. 2006/0292215 discloses methods of using abscisicacid for anti-cancer purposes, and international application No. WO2007/042983 discloses anti-inflammatory activity of abscisic acid.Contents of these patents are incorporated by reference.

Here, Applicants have surprisingly discovered that abscisic acid, and/orsalts, derivatives and analogs thereof, have nutraceutical properties inanimals.

SUMMARY OF THE INVENTION

The present invention is generally directed to compositions comprisingabscisic acid, and/or salts, derivatives and analogs thereof(collectively referred to as “ABA” herein), of which (S)-(+)-abscisicacid is one enantiomer (hereinafter “S-ABA”), and methods of their useas nutraceuticals. Applicants found that compositions of ABA can be usedto treat various ailments, and may also be used as nutraceuticals.

Compositions of the present invention generally comprise ABA. Othercomponents which enhance the biological activity of the ABA mayoptionally be included.

Another embodiment of the present invention is directed to treatment fordiabetes including methods of reducing glucose levels and methods ofdecreasing triglyceride levels in blood comprising administering to ananimal a therapeutically effective amount of ABA.

In yet another embodiment, the present invention is directed toreproduction and more specifically to methods of increasing post-natalsurvival rates and weight comprising administering to an animal in needthereof, or lactating parent of animal in need thereof, atherapeutically effective amount of ABA.

Another embodiment of the present invention is directed to methods ofincreasing immunological function comprising administering to an animalin need thereof a therapeutically effective amount of ABA. Thisimmunological effect may be, at least in part, indicated via theobserved reduction in size of the spleen, as a number of conditions—frominfections to liver disease and some cancers—can cause an enlargedspleen.

Finally in another embodiment, the present invention is directed toenhancing alertness and the treatment of attention deficit disordercomprising administering to an animal a therapeutically effective amountof ABA.

The disclosed embodiments are simply exemplary embodiments of theinventive concepts disclosed herein and should not be considered aslimiting, unless the claims expressly state otherwise.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed to compositions comprisingABA, of which S-ABA is one enantiomer, and methods of their use asnutraceuticals. Applicants found that compositions of ABA can be used totreat various ailments, as well be used as nutraceuticals, in infantformula or as a food ingredient.

Preferred ABA analogs and derivatives are defined by Structures 1, 2,and 3, wherein for Structure 1:

-   -   the bond at the 2-position of the side chain is a cis- or        trans-double bond,    -   the bond at the 4-position of the side chain is a trans-double        bond or a triple bond,    -   the stereochemistry of the alcoholic hydroxyl group is S-, R- or        an R,S-mixture,    -   the stereochemistry of the R1 group is in a cis-relationship to        the alcoholic hydroxyl group,    -   R₁=ethynyl, ethenyl, cyclopropyl, or trifluoromethyl, and    -   R₂=hydrogen or lower alkyl    -   wherein lower alkyl is defined as an alkyl group containing 1 to        4 carbon atoms in a straight or branched chain, which may        comprise zero or one ring or double bond when 3 or more carbon        atoms are present.

For Structure 2:

-   -   the bond at the 2-position of the side chain is a cis- or        trans-double bond,    -   the bond at the 4-position of the side chain is a triple bond,    -   the stereochemistry of the alcoholic hydroxyl group is S-, R- or        an R,S-mixture, R₁=hydrogen or lower alkyl    -   wherein lower alkyl is defined as an alkyl group containing 1 to        4 carbon atoms in a straight or branched chain, which may        comprise zero or one ring or double bond when 3 or more carbon        atoms are present.

For Structure 3:

-   -   the bond at the 2-position of the side chain is a cis- or        trans-double bond,    -   the bond at the 4-position of the side chain is a trans-double        bond,    -   the stereochemistry of the alcoholic hydroxyl group is S-, R- or        an R,S-mixture, R₁=hydrogen or lower alkyl    -   wherein lower alkyl is defined as an alkyl group containing 1 to        4 carbon atoms in a straight or branched chain, which may        comprise zero or one ring or double bond when 3 or more carbon        atoms are present.

Salts of the above analogs including sodium and potassium salts may beused in this invention.

S-ABA is the preferred compound of the compositions and uses herein andhas the structure as follows:

Compositions and methods of the inventions encompass all isomeric formsof the described abscisic acids, their racemic mixtures, enol forms,solvated and unsolvated forms, analogs, prodrugs, derivatives, includingbut not limited to esters and ethers, and acceptable salts. Examples ofsuitable salts that can be used include inorganic salts such as theammonium, lithium, sodium, potassium, magnesium, and potassium salts andorganic amine salts such as the triethanolamine, diethanolamine,dimethylethanolamine, and ethanolamine salts. In one embodiment, theorganic amine salt is the triethanolamine salt. In another embodiment,the organic amine salt is the dimethylethanolamine salt. In yet anotherembodiment, the organic amine salt is the ethanolamine salt. Theseexamples of salts are not limiting as other salts may also be suitablefor use in the present invention. One presently preferred salt is theammonium salt. Other preferred salts are the sodium and potassium salts.The salts may be prepared by contacting the acid form with a sufficientamount of the desired base to produce a salt in the conventional manner.The free acid forms may be regenerated by treating the salt with asuitable dilute aqueous acid solution such as dilute aqueous sulfuric,hydrochloric or phosphoric acid. The free acid forms differ from theirrespective salt forms somewhat in certain physical properties, such astheir solubilities in polar solvents, but the salts are equivalent totheir respective free acid forms for purposes of the invention. (See,for example S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci.,66: 1-19 (1977) which is incorporated herein by reference).

Definitions

The terms “preventing” and “prevention” refer to prophylactic use toreduce the likelihood of a disease, disorder, or condition to which suchterm applies, or one or more symptoms of such disease, disorder, orcondition. It is not necessary to achieve a 100% likelihood ofprevention; it is sufficient to achieve at least a partial effect ofreducing the risk of acquiring such disease, disorder, or condition.

The term “Animal” refer to any animal, or offspring of animal, who isreceiving treatment, is in need of treatment, is taking or receivingtreatment for prevention purposes, and/or is being administered thecomposition. The term “offspring” refers to progeny or descendants ofanimal, and includes born progeny, fetuses and embryos. “Animals” shallinclude but not be limited to poultry, including chickens, whichincludes boilers and layers and male and female breeding stock, geese,duck, turkey, pheasant, cornish hens, swine, cattle, which includes beefand dairy production, sheep, and goats. Further, “Animals” shall alsoinclude catfish, carp, tilapia, trout, crayfish, shrimp, lobster, crab,aquatic mammals, salmon, and white fish.

The term “composition” includes a product comprising ABA (and in thespecified amounts, if indicated), including products with exogenous orupregulated ABA, as well as any product which results, directly orindirectly, from combination of ABA with specified ingredients in thespecified amounts.

The term “administering” or “administration” includes any means forintroducing the ABA of the invention and other therapeutic agents, intothe body, preferably into the systemic circulation. Examples include butare not limited to oral, buccal, sublingual, pulmonary, ophthalmic,transdermal, transmucosal, intranasal, as well as subcutaneous,intraperitoneal, intravenous, intramuscular injection, transplacentaltransfer and lactation.

The term “therapeutically effective amount” means an amount of acompound that, when administered to an animal for treating a disease,condition or attaining a desired result, is sufficient to effect suchtreatment for the disease or desired result. The “therapeuticallyeffective amount” will vary depending on the compound, the disease statebeing treated or health benefit desired, the severity or the diseasetreated, the result desired, the age and relative health of the animal,the route and form of administration, the judgment of the attendingpractitioner, or person attending or caring for animal, and otherfactors. The amount of ABA that is “effective” will vary fromcomposition to composition, depending on the particular use, theparticular ABA, salts, derivatives and analogs thereof, and the like.Thus, it is not always possible to specify an exact “effective amount.”However, an appropriate “effective amount” in any individual case may bedetermined by one of ordinary skill in the art using routineexperimentation.

The term “treating” and “treatment” have a commonly understood meaningof administration of a remedy to an animal, or the animal's parent, whohas or is suspected of having a disease or a condition, and refer toreversing, alleviating, inhibiting, or slowing the progress of thedisease, disorder, or condition to which such terms apply, or one ormore symptoms of such disease, disorder, or condition, or preventing ordecreasing the chances of a disease, condition, disorder or outcome fromoccurring, or to increase effects of a specified physiological responseor health benefit.

As used herein, the terms “reducing”, “suppressing” and “inhibiting”have their commonly understood meaning of lessening or decreasing. Asused herein, the term “progression” means increasing in scope orseverity, advancing, growing or becoming worse. As used herein, the term“recurrence” means the return of a pre-treatment state after a period ofremission.

As used herein, the term “nutraceutical” is commonly understood to meanany substance containing ABA that is a food or liquid, part of a food orliquid, or addition to food or liquid, and that provides medical orhealth benefits, including the prevention and treatment of disease, orthat triggers a physiological response independent or in excess of asubstance that does not contain exogenous or upregulated ABA. Suchproducts may range from isolated nutrients, dietary supplements,specific diets, genetically engineered designer foods, herbal products,and processed foods such as cereals, soups, nutritional bars, beverages,tablets, capsules, solutions, emulsions, bars, gels, shakes, yogurts,breads, juices, and other nutraceuticals.

As used herein, all numerical values relating to amounts, weightpercentages and the like are defined as “about” or “approximately” eachparticular value, namely, plus or minus 10%. For example, the phrase “atleast 5% by weight” is to be understood as “at least 4.5% to 5.5% byweight.” Therefore, amounts within 10% of the claimed values areencompassed by the scope of the claims.

Compositions for Administration

In some embodiments, the compositions of the present invention can beincluded in a suitable vehicle suitable for oral ingestion. Suitableacceptable carriers include solid fillers or diluents and sterileaqueous or organic solutions. The active compound is present in suchcompositions in an amount sufficient to provide the desired effect.

Compositions contemplated for use in the practice of the presentinvention can be used in the form of a solid, a solution, an emulsion, adispersion, a micelle, a liposome, and the like, wherein the resultingcomposition contains one or more of the active ingredients in admixturewith an organic or inorganic carrier or excipient suitable for nasal,enteral, or parenteral applications.

The active ingredients may be combined, for example, with the usualnon-toxic, physiologically acceptable carriers for tablets, pellets,capsules, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, suppositories, solutions, emulsions, suspensions,hard or soft capsules, caplets or syrups or elixirs and any other formsuitable for use. The possible carriers include glucose, lactose, gumacacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc,corn starch, keratin, colloidal silica, potato starch, urea, mediumchain length triglycerides, dextrans, and other carriers suitable foruse in manufacturing preparations, in solid, semisolid, or liquid form.In addition auxiliary, stabilizing, thickening and coloring agents maybe used.

In another embodiment, the compositions of the present invention may beformulated for an intranasal, intravenous, transdermal or opthalmicadministration. It is within a skill in the art to formulate thecompositions for such administration.

In a different embodiment, the compositions of the present invention maybe formulated with foodstuffs for oral consumption. In anotherembodiment, the composition of the present invention may be formulatedas a nutritional supplement for consumption, for example as a solid orliquid, such as a tablet, capsule, solution, emulsion, bar, gel, shakeor the like. In other embodiments, the compositions of the presentinvention may be formulated with yogurts, cereals, breads, juices andother nutraceuticals. In more embodiments, the compositions of thepresent invention may be incorporated with yogurts, cereals, breads,juices and other nutraceuticals including, but not limited to,foodstuffs which provide health benefits.

In a different embodiment, the compositions of the present invention maybe formulated in a liquid composition for oral consumption. It is withina skill in the art to formulate the compositions in liquid compositionsfor oral consumption.

In other embodiments, the compositions of the present invention may beadministered through a lactating parent to offspring, throughtransplacental transfer from parent to offspring, by IV, or in an infantformula for the offspring.

Diseases and Conditions to Be Treated With Compositions and Methods ofthe Invention

The invention provides methods of treating and/or preventing a diseaseor condition comprising administering to an animal in need thereof atherapeutically effective amount of the compositions of the invention.

In another embodiment, the present invention is directed to reproductionand more specifically to methods of increasing post-natal survival ratesand weight comprising administering to an animal in need thereof atherapeutically effective amount of ABA.

Another embodiment of the present invention is directed to methods ofincreasing immunological function comprising administering to an animalor animal in need thereof a therapeutically effective amount of ABA.This may result at least in part due to the reduction in size of thespleen in the animal. The spleen is an organ which plays a role infiltering and recycling blood while platelets and white cells are storedthere, all playing a role with the immune system.

A preferred range of a therapeutically effective amount of ABA for thevarious methods is from about 0.1 mg/kg/day to about 1000 mg/kg/day. Amore preferred range of a therapeutically effective amount of ABA isfrom about 10 mg/kg/day to about 1000 mg/kg/day. An especially preferredrange of a therapeutically effective amount of ABA is from about 50mg/kg/day to about 500 mg/kg/day. An especially preferred range of atherapeutically effective amount of ABA is from about 50 mg/kg/day toabout 200 mg/kg/day.

The preferred composition comprises S-ABA.

One embodiment of the present invention is liquid compositions that canbe prepared as either ready-to-use dilutions or dilutable concentrates.The embodiment of the present invention can be a solution containingfrom 0.5% to as much as 50% by weight of ABA. The dilutable concentratescan be diluted into water directly to a final application concentrationor to any intermediate dilution, without risk of precipitation of theactive ingredient. The aqueous formulations according one embodiment ofthe present invention are inexpensive to manufacture, safe to handle anduse, and the ABA active ingredient is stable under storage and shippingconditions. A person having ordinary skill in the art would be able todetermine how to prepare the final aqueous solution concentration fordirect application to animals without undue experimentation, without anychance of causing precipitation of the active ingredient, and withoutlong and laborious stirring to bring the active ingredient intosolution.

Compositions of the present invention may be prepared as a single unitdose or as a plurality of single unit doses. As used herein, a “unitdose” means a discrete amount of the composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientthat would be administered to an animal or a fraction thereof.

Compositions of the present invention may be liquids or lyophilized orotherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween® (Tween is a registered trademark of UniqemaAmericas, LLC) 20™, Tween® 80™, Pluronic® (Pluronic is a registeredtrademark of BASF Corporation) F68™, bile acid salts), solubilizingagents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g.,ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal™,benzyl alcohol, parabens), bulking substances or tonicity modifiers(e.g., lactose, mannitol), covalent attachment of polymers such aspolyethylene glycol to the protein, complexation with metal ions, orincorporation of the material into or onto particulate preparations ofpolymeric compounds such as polylactic acid, polyglycolic acid,hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellaror multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance. Controlled orsustained release compositions include formulation in lipophilic depots(e.g., fatty acids, waxes, oils).

Further, as used herein acceptable carriers are well known to thoseskilled in the art and include, but are not limited to, 0.01-0.1 M andpreferably 0.05M phosphate buffer or 0.9% saline. Additionally, suchacceptable carriers may be aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media.

Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's and fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

Controlled or sustained release compositions according to the inventioninclude formulation in lipophilic depots (e.g. fatty acids, waxes,oils). Also comprehended by the invention are particulate compositionscoated with polymers (e.g. poloxamers or poloxamines) and the compoundcoupled to antibodies directed against tissue-specific receptors,ligands or antigens or coupled to ligands of tissue-specific receptors.Other embodiments of the compositions according to the inventionincorporate particulate forms, protective coatings, protease inhibitorsor permeation enhancers for various routes of administration, includingparenteral, pulmonary, nasal and oral.

Compounds modified by the covalent attachment of water-soluble polymerssuch as polyethylene glycol, copolymers of polyethylene glycol andpolypropylene glycol, carboxymethyl cellulose, dextran, polyvinylalcohol, polyvinylpyrrolidone or polyproline are known to exhibitsubstantially longer half-lives in blood following intravenous injectionthan do the corresponding unmodified compounds. Such modifications mayalso increase the compound's solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. As a result, the desired in vivo biological activity may beachieved by the administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

The preparation can comprise the ABA, alone, or can further include apharmaceutically acceptable carrier, and can be in solid or liquid formsuch as tablets, powders, capsules, pellets, solutions, suspensions,elixirs, emulsions, sprays, gels, creams, or suppositories, includingrectal and urethral suppositories. Pharmaceutically acceptable carriersinclude gums, starches, sugars, cellulosic materials, and mixturesthereof. The preparation containing the ABA can be administered to ananimal by, for example, subcutaneous implantation of a pellet. Thepreparation can also be administered by intranasal, intravenous,intraarterial, or intramuscular injection of a liquid preparation.Administration can also be accomplished by use of a rectal suppositoryor a urethral suppository.

The preparations administrable by the invention can be prepared by knowndissolving, mixing, granulating, or tablet-forming processes. For oraladministration, ABA and the like are mixed with additives customary forthis purpose, such as vehicles, stabilizers, or inert diluents, andconverted by customary methods into suitable forms for administration,such as tablets, coated tablets, hard or soft gelatin capsules, aqueous,alcoholic or oily solutions. Examples of suitable inert vehicles areconventional tablet bases such as lactose, sucrose, or cornstarch incombination with binders such as acacia, cornstarch, gelatin, withdisintegrating agents such as cornstarch, potato starch, alginic acid,or with a lubricant such as stearic acid or magnesium stearate.

Examples of suitable oily vehicles or solvents are vegetable or animaloils such as sunflower oil or fish-liver oil. Preparations can beeffected both as dry and as wet granules. For parenteral administration(subcutaneous, intravenous, intra-arterial, or intramuscular injection),the ABA and the like are converted into a solution, suspension, oremulsion, if desired with the substances customary and suitable for thispurpose, for example, solubilizers or other auxiliaries. Examples aresterile liquids such as water and oils, with or without the addition ofa surfactant and other pharmaceutically acceptable adjuvants.Illustrative oils are those of petroleum, animal, vegetable, orsynthetic origin, for example, peanut oil, soybean oil, or mineral oil.In general, water, saline, aqueous dextrose and related sugar solutions,and glycols such as propylene glycols or polyethylene glycol arepreferred liquid carriers, particularly for injectable solutions.

The preparation of compositions which contain an active component iswell understood in the art. Such compositions may be prepared asinjectables, either as liquid solutions or suspensions; however, solidforms suitable for solution in, or suspension in, liquid prior toinjection can also be prepared. The preparation can also be emulsified.Active therapeutic ingredients are often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like or any combination thereof.

In addition, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentswhich enhance the effectiveness of the active ingredient.

Animal Feed

The composition of the invention may preferably take the form of animalfeed and includes protein, fat, fiber, calcium, and phosphorous. Apreferred feed would include corn and/or wheat, soybean meal, fat,animal by-product, meat and bone meal, and vitamins and minerals.

ABA can be initially mixed into a premix. The term “premix” is intendedto mean a feed composition that is prepared as an initial mix containingthe active ingredient and, for example, a carrier, and is then blendedinto the final feed. In the present invention, it is generally suggestedthat, for ease of calculation and use, the premix is blended with aboutone metric ton (MT) of conventional feed, with the result that thenecessary dosage requirements of the ABA are provided to the animals.When preparing one metric ton (MT) of finished feed, the premix of theinvention preferably comprises about 1 to 2000, and more preferably,about 5 to 500, grams of ABA. Carriers for use in a premix are wellknown by those having skill in the art, and appropriate concentrationscan be readily determined.

The ABA may be added to the carrier as a dry powder or as a liquidsolution or suspension. When added as a liquid, the ABA may be dissolvedor suspended in a liquid with stirring at room temperature. Such liquidmay be water or a suitable solvent or another product used for animalfeed that is already in liquid form. Because of ABA's liquid solubilitycharacteristics, it may form a suspension. A predetermined amount of ABAis then added to the conventional premix, and will not overly wet it.Once the premix is prepared, the premix is then added to the final feed,preferably at a rate in the range of one quarter of a pound (lb.) tofive pounds (lbs.) of premix to one metric ton (MT) of feed, to supplydaily requirements of the ABA for the poultry.

The ABA solid or solution or liquid suspension can be added directly tothe premix material, and then mixed. Mixing can be accomplished by anyknown means, such as by a standard horizontal or vertical blender.Mixing time will again vary depending upon the particular ingredients ofthe premix, and can take as long as is necessary to assure that theingredients are thoroughly mixed.

The premix is then incorporated into the feed to be fed to the poultry.In a more preferred embodiment, ABA is blended with the carrier to formthe premix, and the premix is directly blended into the final feed.While there is no evidence that use of the higher amounts would causeany toxicity problems in treated poultry, it will affect costconsiderations. Because the premix generally will be added to one-toncharges of feed (as is common in the industry), the correspondencebetween one gram of ABA added to the premix yields about 1 ppmconcentration of ABA in the feed. Thus, 5 grams of ABA added to onepound of premix, which in turn is added to one metric ton (MT) of feed,yields about a 5 ppm effective ABA concentration.

While the ABA can be mixed with a premix material prior to incorporationinto the finished feed, the appropriate amount of ABA may be directlyblended into or sprayed upon the feed. The preferred additive range ofABA in finished feed, whether added directly or via a premix, is about 1to 2000 grams per metric ton (MT) of feed; more preferably about 5 to500 grams per metric ton (MT) of feed.

Feed is conventionally prepared in a large bin or mixer in which thefeed ingredients are added in descending weight order according to theirprevalence in the ultimate feed mixture. Thus, cracked or ground grainwould be the primary ingredient. Minor ingredients are then added.Micro-ingredients are added last. These include vitamins, drugs, growthpromoters, antibiotics, and, in the present case, ABA. Thus, ABA can beone of the micro-ingredients and is added to the feed in the finalblending step. The feed is blended for conventional time periods.

The feed comprising the ABA is fed to livestock and fish in standardform, such as a mash, crumble or pellet, and at standard feed dosageranges and rates.

Another embodiment of the present invention is liquid compositions thatcan be prepared as either ready-to-use dilutions or dilutableconcentrates. The embodiment of the present invention can be a solutioncontaining from 0.5% to as much as 50% by weight of ABA. The dilutableconcentrates can be diluted into water directly to a final applicationconcentration or to any intermediate dilution, without risk ofprecipitation of the active ingredient. The aqueous formulationsaccording to one embodiment of the present invention are inexpensive tomanufacture, safe to handle and use, and the ABA active ingredient isstable under storage and shipping conditions. A person having ordinaryskill in the art would be able to determine how to prepare the finalaqueous solution concentration for direct application to animals withoutundue experimentation, without any chance of causing precipitation ofthe active ingredient, and without long and laborious stirring to bringthe active ingredient into solution.

Another embodiment of the present invention is an ABA water solutionthat serves as a drinking source of water for the animals. Suchsupplemented water solution could be prepared by dissolving dry powderABA in drinking water or by using a liquid solution or suspensionconcentrate of ABA. The preferred additive range of ABA in drinkingwater is about 1 to 2000 parts per million (ppm) or about 1 to 2000milligrams of ABA per liter of water; more preferably about 5 to 500parts per million (ppm) or about 5 to 500 milligrams of ABA per liter ofwater. A person having ordinary skill in the art would be able todetermine how to prepare the final aqueous solution for directapplication to animals without undue experimentation, without any chanceof causing precipitation of the active ingredient, and without long andlaborious stirring to bring the active ingredient into solution.

In another embodiment of the present invention ABA can be applieddirectly onto animal feed once it has been prepared. For example, as canbe the practice with some enzymes, the ABA can be applied directly tothe finished feed. In a preferred embodiment, an aqueous solution of ABAis sprayed onto the finished feed in its final form, such as a pellet,prior to delivering the feed to the animals.

The advantageous properties of this invention can be observed byreference to the following examples that illustrate the invention. Theseexamples are provided for the purposes of illustration and are notintended to limit the scope of the present invention.

EXAMPLES

Tests were conducted with (S)-(+)-abscisic acid in accordance withstandard guidelines and procedures as evidenced by EPA SubchronicToxicity Test Guidelines: 870.3050-Repeated Dose 28-Day Oral ToxicityStudy in Rodents (July 2000); 870.3100-90-Day Oral Toxicity in Rodents(August 1998); 870.3650-Combined Repeated Dose Toxicity Study with theReproduction/Developmental Toxicity Screening Test (July 2000); and870.3200-21/28-Day Dermal Toxicity (August 1998), as well asOrganization for Economic Co-operation and Development (OECD) Guidelinesfor the Testing of Chemicals including: Test No. 407: Repeated Dose28-day Oral Toxicity Study in Rodents; Test No. 408: Repeated Dose90-Day Oral Toxicity Study in Rodents; Test No. 416: Two-GenerationReproduction Toxicity; and Test No. 410: Repeated Dose Dermal Toxicity:21/28-day Study.

Examples of the data from these studies are presented below.

Two Generation Reproduction Toxicity Study

This study was conducted to determine the potential adverse effects ofthe test substance on reproduction in a 2-generation study. Thisincluded determining the effects of the test substance on male andfemale reproductive processes, including gonadal function, estrouscyclicity, mating behavior, conception, gestation, parturition,lactation, weaning, and on growth and development of the offspring. Aminimum of 1 litter was produced in each generation.

Three groups of male and female were offered ABA, continuously in thediet for at least 70 consecutive days prior to mating. Target testssubstance concentrations were 10,000, 15,000, and 20,000 ppm for the F0and F1 generations. A concurrent control group of 30 rats/sex wasoffered the basal diet continuously throughout the study. FO animalswere approximately 7 weeks of age at the initiation of test dietadministration. The test diet was administered to the offspring selectedto become the F1 generation following weaning. The F0 and F1 malescontinued to receive the test substance throughout mating and continuingthrough the day prior to euthanasia. The F0 and F1 females continued toreceive the test substance throughout mating, gestation, and lactation,and through the day of euthanasia. For both generations (F1 and F2), 8pups per litter (4 per sex, when possible) were selected to reduce thevariability among the litters. Offspring (30/sex/group, if possible)from the pairing of the F0 animals were selected to constitute the F1generation. F0 males and females were exposed for 127-130 consecutivedays and F1 males and females were exposed for 178-186 consecutive days.

All animals were observed twice daily for appearance and behavior.Clinical observations, body weights, and food consumption were recordedat appropriate intervals for males throughout the study and for femalesprior to mating and during gestation and lactation. All F0 and F1females were allowed to deliver and rear their pups until weaning onlactation day 21. Clinical observations, body weights, and sexes for F1and F2 pups were recorded at appropriate intervals. Nonselected F1 pupsand all surviving F2 pups were necropsied. Selected organs were weighedfrom 1 pup/sex/litter from F1 and F2 pups that wee necropsied. Eachsurviving F0 and F1 parental animal received a complete detailed grossnecropsy following the completion of weaning of the F1 and F2 pups,respectively; selected organs were weighed. Spermatogenic endpoints wererecorded for all F0 and F1 males, and ovarian primordial follicle countswere recorded for all F0 and F1 females in the control and high-exposuregroups and all F0 and F1 females suspected of reduced fertility.Designated tissues from all F0 and F1 parental animals were examinedmicroscopically.

Reproduction

Table 1 demonstrates increased postnatal survival rates of offspring inresponse to S-ABA treatments.

TABLE 1 POSTNATAL SURVIVAL (% PER LITTER) POSTNATAL DAY 0 BIRTH- 4(RELATIVE 4 (POST- 4 (POST- DOSE TO # 1-4 (PRE- SELECTION)- (PRE-SELECT)- (PPM) BORN) 0-1 SELECTION) 7 7-14 14-21 SELECT) 21    0 MEAN98.4 98.0 99.5 98.6 99.5 100.0 95.9 98.1 N 26 26 26 26 26 26 26 2610,000 MEAN 99.7 100.0 98.9 99.5 100.0 100.0 98.6 99.5 N 25 25 25 25 2524 25 24 15,000 MEAN 96.1 98.5 98.5 99.6 98.7 100.0 93.2 98.2 N 29 28 2828 28 28 29 28 20,000 MEAN 98.5 99.7 98.5 98.5 99.5 100.0 96.8 98.0 N 2525 25 25 25 25 25 25

As a result of this test data, we see that S-ABA increases postnataloffspring survival rates. For instance, by days 4 through 7, a 10,000ppm dose resulted in an increase in mean survival from 98.6% to 99.5% inrats.

Table 2 demonstrates increased weight gain of offspring in response toS-ABA treatments.

TABLE 2 OFFSPRING WEIGHT (grams) DOSE POSTNATAL DAY (PPM) 1 4 7 14 21   0 MALE MEAN 6.9 9.1 14.0 29.7 46.8 N 26 26 26 26 26 FEMALE MEAN 6.58.5 13.0 28.2 44.4 N 26 26 26 26 26 10,000 MALE MEAN 7.1 9.7 15.2 30.947.9 N 25 25 25 25 24 FEMALE MEAN 6.6 9.1 14.3 29.9 45.5 N 25 25 25 2524 15,000 MALE MEAN 6.9 9.3 14.7 30.8 47.8 N 28 28 28 28 28 FEMALE MEAN6.6 8.8 14.0 29.8 45.8 N 28 28 28 28 28 20,000 MALE MEAN 7.0 9.3 15.131.2 48.1 N 25 25 25 25 25 FEMALE MEAN 6.6 8.8 14.5 30.5 46.6 N 24 24 2424 24

As a result of this test data, we see that S-ABA increases offspringweight gain. For instance, by day 21, a 10,000 ppm dose resulted in anincrease in mean offspring weight from 46.8 grams to 47.9 grams in malerats. The corresponding increase in mean offspring weight for femalerats was from 44.4 grams to 45.5 grams.

This result is also suggestive that ABA could be substituted forprophylactic antibiotics that are often utilized in animal reproduction.

Table 3 demonstrates decreased offspring mortality levels in response toS-ABA treatments.

TABLE 3 DOSE (PPM) Offspring Born FOUND DEAD 0 328 15 10,000 322 415,000 375 15 20,000 327 11

As a result of this test data, we see that S-ABA decreases offspringmortality. For instance, a 10,000 ppm dose resulted in a decrease from4.57% to 1.24%.

Table 4 demonstrates increased mean live offspring per litter inresponse to S-ABA treatments.

TABLE 4 Dose (PPM) 0 10,000 15,000 20,000 Mean Live 11.8 12.3 13.1 12.3Offspring Born 25 27 27 27 N

As a result of this test data, we see that S-ABA increases the number oflive offspring per litter. For instance, a 15,000 ppm dose resulted inan increase in mean offspring number from 11.8 to 13.1 per litter.

Further supporting data can be seen below in Tables 2 and 3 showing thatat necropsy fetus numbers and follicle size increase with the additionof S-ABA.

TABLE 5 FETAL DATA AT SCHEDULED NECROPSY SEX GROUP MALE FEMALE VIABLEFETUSES 1 N 154 179 343 0 MG/KG/DAY MEAN 5.5 7.2 13.7 2 N 187 181 368500 MG/KG/DAY MEAN 7.5 7.2 14.7 3 N 188 166 354 750 MG/KG/DAY MEAN 7.56.6 14.2 4 N 170 208 378 1,000 MG/KG/DAY MEAN 6.8 8.3 15.1

TABLE 6 OVARIAN PRIMORDIAL FOLLICLE COUNTS FEMALES GROUP 0 PPM 10,000PPM 15,000 PPM 20,000 PPM PRIMORDIAL FOLLICLES MEAN 120.1 257.5 214.0270.3 N 30 5 1 30

Immune System Health

Two-generation reproductive toxicity studies of S-ABA in ratsdemonstrated spleen weight reduction in both males and second generationpups as seen in Table 7 and 8.

TABLE 7 MALES GROUP 0 PPM 10,000 PPM 15,000 PPM 20,000 PPM SPLEEN WEIGHT(GRAMS) MEAN 1.00 0.98 0.97 0.92 % DIFFERENCE −2.0 −3.0 −8.9 N 30 30 2830

TABLE 8 SUMMARY OF ORGAN WEIGHTS AND RELATIVE ORGAN WEIGHTS SECONDGENERATION PUPS GROUP 0 PPM 10,000 PPM 15,000 PPM 20,000 PPM SPLEENWEIGHT (GRAMS) MEAN 0.2334 0.1989 0.1918 0.1979 % DIFFERENCE −14.8 −17.8−15.2 N 25 25 27 27

Reduced spleen size, as shown in the tables/data above, may be directlyrelated to improved health and improved mortality shown in theReproduction tables/data above.

What is claimed is:
 1. A method of improving the productivity of animalreproduction comprising administering to a pregnant animal in needthereof a therapeutically effective amount of abscisic acid (ABA),wherein the ABA is administered in or with an animal feed.